The invention relates to a method of virus manipulation; means therefor and products thereof which have particular, but not exclusive, application in gene therapy/vaccine development.
A virus-derived vector capable of efficient gene delivery to human T lymphocytes would have a wide range of uses in human gene therapy. An obvious disease target would be human immunodeficiency virus (HIV) infection, where such a vector could have use both in the prevention and therapy of infection. Two virus vector systems which do not cause a cytopathic effect in vitro, retroviruses and adeno-associated virus, both target dividing cells only, and are thus inappropriate for use with a CD4 T cell population which are mainly non-dividing.
Virus based gene therapy systems currently in clinical trial or in development include vectors based on adenoviruses, retroviruses and human herpesviruses (1, 2, 3). All these systems have inherent disadvantages. Adenovirus vectors have constraints on the size of heterologous DNA incorporated, can cause toxic side effects and induce a vigorous immune response resulting in rapid clearance of infected and therefore gene targeted cells (4, 5). A major drawback in retroviral systems based on murine leukemia viruses is their inability to infect non-dividing cells. Thus cells must be removed, activated, infected in vitro, and then delivered back to the patient. A further disadvantage is the high inherent mutation rate caused by reverse transcription (6).
Herpesvirus vector systems offer the potential of delivering  greater than 50 kb of heterologous DNA, the infection of non-dividing cells and maintenance of their genome episomally in a non-replicative form (7). However, nearly all vector systems in development to date are based on herpes simplex virus and are likely to be ineffective in many individuals due to an immune response present in  greater than 80% of the population, already induced by the wild type virus (8). A herpesvirus of non-human origin, capable of infecting human cells, therefore represents an attractive candidate as a gene therapy vector, as there will be no innate immune response in the recipient to prevent infection in vitro.
Herpesvirus saimiri (HVS) is a lymphotropic rhadinovirus (xcex32 herpesvirus) of squirrel monkeys (Saimiri sciureus). The virus genome may be detected in an episomal form in T cells and causes no apparent disease in the natural host. Whereas type A and B strains similarly do not cause apparent disease in other monkey species, C type strains of this virus are oncogenic in certain New World primates (9). C strains also have the ability to transform human T cells in vitro. The gene product responsible for cell transformation has been identified as the STP gene (ORF1) (10). STP is non-essential for virus replication in vitro and in vivo; natural deletion mutants exist in C strains which are non-oncogenic. Therefore a virus of strain A, which has the STP gene deleted is unable to transform any type of cell. Virus strains lacking this gene and carrying several heterologous genes have been constructed and studies carried out in vitro have demonstrated high efficiency and long term expression of the heterologous gene product (11). The virus DNA remains episomal with no detectable expression of virus genes, but with stable heterologous expression in the absence of selection. Furthermore, the virus genome segregates efficiently between dividing cells, presumably in a manner similar to the human xcex32 herpesvirus, Epstein-Barr Virus. Advantageously, HVS which naturally infects non-human primates has also been found to infect human T lymphocytes. As previously mentioned, this feature of HVS can be used to advantage for providing in man a wide range of gene therapies. For example, it is of note that a major target cell type for HIV infection in man is T lymphocytes. We therefore speculated that the expression of HIV protein in the correct oligomeric configuration in vivo on the surface of a T lymphocyte should induce an effective humoral and cellular anti-HIV immune response. HVS can be used to advantage in the delivery of a gene encoding large HIV proteins because, as previously mentioned, it has the potential of delivering  greater than 50 kb of heterologous DNA and is further capable of infecting T lymphocytes.
In our studies we chose an envelope gene from a primary isolate of HIV as the gene to be delivered by HVS for the development of the potential vaccine.
Studies have shown that immunogens based on the envelope protein or defined epitopes thereof induce both virus neutralizing antibodies and HIV-specific cytotoxic T lymphocytes (12, 13, 14, 15). Furthermore, chimpanzees have been successfully protected against HIV challenge, with protection correlated with the presence of high titre neutralizing antibodies (12). However, recent studies have shown that antibodies raised against these immunogens encoding envelope sequences from laboratory adapted isolates are ineffective at neutralizing primary isolates from HIV infected individuals (16). Envelope proteins from primary isolates confer different characteristics to the virus as compared to laboratory adapted isolates. These include the ability to infect macrophages and the inability to induce syncytia, the cytopathic effect seen with laboratory adapted isolates. Furthermore, anti-envelope antibodies from HIV positive individuals show cross isolate neutralizing ability (17, 18). Analysis of HIV neutralizing antibodies have also suggested that the envelope immunogen must be presented in the authentic, oligomeric, non-denatured form to induce heterologous neutralizing antibodies (15, 19). Recent reports also indicate the need to elicit a polyclonal antibody response as human monoclonal antibodies isolated from patients fail to neutralize certain primary isolates (18, 20). Further evidence for the feasibility of a vaccine strategy utilizing a primary macrophage-tropic envelope is provided by a study using the simian immunodeficiency model (SIV) model (21). Macaques immunized with an attenuated macrophage-tropic SIV developed cross-neutralizing antibodies and were successfully protected against heterologous isolate challenge.
The studies outlined above indicate that the induction of a vigorous immune response against the HIV envelope protein may protect against virus challenge or constrain the virus if infection does occur. Long term survivors who remain health); 12-15 years after infection have high levels of neutralizing antibodies (17). Delivery of a primary isolate envelope gene by a virus based vector to the correct cell population offers the potential to induce an effective anti-HIV cross-isolate immune response, an essential requirement of any candidate HIV vaccine. This gene therapy approach may also have therapeutic value to individuals who have progressed to AIDS. It has been shown that expression of envelope protein in T cells induces partial resistance to infection, and significantly induces total resistance to cytopathic effects (22). T cells infected with the recombinant HVS may therefore be protected in vivo.
We believe that vectors derived from HVS offer a wide range of opportunities to target both protective and therapeutic genes to human T cells, offering the unique opportunity to deliver a stably-expressing, extrachromosmal element, to a non-dividing cell population. Moreover we have demonstrated efficient infection of the T-cell line Jurkat, by a recombinant HVS vector expressing HIV gp160.
It is therefore a first object of the invention to provide a gene delivery system/vaccine to deliver at least a part of at least one preselected gene to a specific cell population in which the encoded heterologous protein is presented as an antigen.
In its broadest aspect the invention concerns the use of HVS to deliver heterologous genetic material to a specific cell population and ideally to T lymphocytes and/or macrophages.
According to a first aspect of the invention there is therefore provided a herpesvirus saimiri vector which has inserted therein at least a part of a gene encoding an envelope protein of HIV for use in delivering said protein to a specific cell population.
Preferably, the gene encoding an envelope protein of HIV is from a primary isolate of HIV.
Preferably, the specific cell population is T lymphocytes and/or macrophages.
In yet a further preferred embodiment of the invention said HVS lacks or has a mutation in its STP gene (ORF1) such that the gene product is lacking or is non-functional.
HVS may be suitably modified in order to facilitate its use as an agent for effecting gene transfer. For example, non-essential genes may be deleted in order to facilitate the delivery of large amounts of heterologous DNA, that is in the order of  greater than 50 kb. In addition, or alternatively HVS may be modified so as to render it safe and controllable, for example, genes encoding transcriptional control proteins may be mutated and/or deleted so as to disable the replication cycle of the virus.
In our co-pending application, UK patent application no. 9521711.3 we have described in detail the way in which HVS can be modified to advantage to provide for a suitable gene delivery agent and we now incorporate herein by way of reference the content of this co-pending application on the understanding that features of this co-pending application may be used in combination with any features described herein and may be claimed in combination with any one or more feature described herein.
Thus in a further preferred embodiment of the invention there is provided said HVS, as described above, modified in accordance with any one or more of the features described in UK patent application no. 9521711.3.
Ideally, said HVS is modified so that the genes related to ORF4 and/or ORF15 are mutated and/or deleted. This action is favoured because these genes encode glycoproteins that are thought to down-regulate complement activity and thus they may have an affect which is prejudicial to the success of our delivery system/vaccine.
It will be apparent to those skilled in the art that the nature of the heterologous DNA will be determined having regard to the nature of the protein to be introduced into the specific cell population. Where the specific cell population is a T lymphocyte population and/or macrophages it is advantageous to use heterologous DNA encoding at least a part of a protein which when expressed is presented on the surface of a T lymphocyte and/or macrophage. Thus it is preferred to use heterologous DNA encoding at least a part of an envelope, membrane binding protein or the like. The presentation of such protein on the surface of a T lymphocyte should induce an effective humoral and cellular anti-protein immune response.
Further, the nature of heterologous DNA to be provided in said HVS will be such that the corresponding protein fragment or product is sufficient to produce an acceptable or effective response when expressed in, or on the surface of, a specific cell population.
According to a further aspect of the invention there is provided a target cell including said HVS of the invention.
According to a yet further aspect of the invention there is provided a cell transduced with the HVS of the invention.
According to a yet further aspect of the invention there is provided a vector adapted to introduce heterologous DNA encoding at least a part of an HIV protein to be delivered to a specific, which vector is adapted to insert said heterologous DNA into HVS.
According to a yet further aspect of the invention there is provided a method of delivering selected HIV heterologous genetic material to a specific cell population comprising exposing at least a target cell population to herpesvirus saimiri which includes said preselected HIV heterologous material under conditions which favour infection of said population with said virus.
In yet a further embodiment of the invention there is provided a vaccine comprising said HVS and any other suitable excipient or canier. For example, the vaccine may be in the form of a fluid which may be injected intravenously, intramuscularly or subcutaneously into an individual to be vaccinated. Alternatively, said vaccine may be in the form of a fluid which is adapted for use as an aerosol whereby droplets of the vaccine can be delivered to the respiratory tract of an individual to be vaccinated.
Other conventional means of vaccine delivery may be employed in the working of the invention and will be well known to those skilled in the art.
According to a yet further aspect of the invention there is provided use of a vaccine as herein before described for the prophylaxis or treatment of HIV.
The present invention also provides a method of preventing and/or treating an individual likely to contract or suffering from HIV infection, comprising administering a therapeutically effective amount of the vaccine as herein before described.
Preferably, administration of the vaccine may be repeated at selected time intervals.
According to a yet further aspect of the invention the is provided a pharmaceutical comprising a herpesvirus saimiri vector which has inserted therein at least a part of a gene encoding an envelope protein of HIV and optionally further including any of the preferred features hereinbefore described.
According to a yet further aspect of the invention the is provided use of a herpesvirus saimiri vector which has inserted therein at least a part of a gene encoding an envelope protein of HIV in the manufacture of a medicament for use in preventing and/or treating an individual likely to contract or suffering from HIV infection and optionally further including any of the preferred features hereinbefore described.